Drug-resistance is an important factor of failure to cure patients with high-risk neuroblastoma, and little is known of the contribution of the microenvironment to drug resistance in this cancer. In collaboration, the investigators of this project have demonstrated that bone marrow mesenchymal stem cells (BMMSC) and tumor associated monocytes/macrophages (TAM) share a common pathway of interaction with human neuroblastoma cells that leads to drug resistance. Central to this pathway is IL-6 produced by these tumor cellstimulated stromal cells and its downstream target STATS. We hypothesize that such protection allows tumor cells to survive and to undergo additional genetic and epigenetic alterations that render them even more resistant. We also postulate that combining inhibitors of EMDR pathways like IL-6/IL- 6R/STAT3 with chemotherapy or targeted therapy in patients with high-risk neuroblastoma will improve therapeutic response and decrease disease recurrence with multidrug resistant tumor cells. Aim1 will use co-cultures of drug sensitive neuroblastoma cell lines and BMMSC, pre-osteoblasts, and monocytes/macrophages to determine if they cause resistance to cytotoxic drugs, to identify mechanisms of resistance, and to test the effect of pathway inhibitors in restoring drug sensitivity. Experiments will then be validated with tumor containing bone marrow samples obtained from patients with stage 4 neuroblastoma. Using long term co-cultures of drug sensitive neuroblastoma cell lines in the presence of bone marrow-derived stromal cells we shall test whether EMDR promotes epigenetic changes that will lead to a state of drug resistance that has become independent of the microenvironment. Aim 2 will evaluate EMDR mechanism in vivo using a transgenic model of MYCN-NA high-risk neuroblastoma (NB-Tag) in which key genes involved in lL-6/IL-6R/STAT3-mediated EMDR will be ablated. Aim 3 will test the hypothesis that blocking 1L-6/IL- 6R/STATS pathways responsible for EMDR in neuroblastoma enhances the response to chemotherapy. These experiments initially will test lenalidomide, an immune modulator, and tociluzumab, an anti-IL-6R antibody as well a small inhibitors of SI PRI and JAK2. Project 1 will collaborate with Project 2 in testing whether the SDF1a/CXCR4 plays a role in EMDR in neuroblastoma and with Project 3 in determining the contribution of S1P/S1PR1 to IL-6-mediated EMDR and in testing inhibitors of S1PR1 and JAK2 in preclinical models of neuroblastoma. We anticipate that our research will provide a new paradigm for the initial treatment not only of children with high-risk neuroblastoma but also of patients with other cancers.